Cross-incompatibility traits from teosinte and their use in corn

ABSTRACT

The present invention relates to a teosinte crossing barrier trait. Plants containing said trait exhibit the phenotype of cross-incompatiblity. The present invention also relates to new cross-incompatible plants, including inbred, hybrid, haploid, apomictic and/or genetically engineered plants, containing the teosinte crossing barrier trait and exhibiting commercially desirable characteristics.

CROSS-RELATED APPLICATION INFORMATION

This application claims priority from U.S. Application No. 60/193,082filed on Mar. 30, 2000.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support awarded bythe following agencies: USDA 98-CRHF-0-6055.

TECHNICAL FILED OF THE INVENTION

The present invention relates to a teosinte crossing barrier trait.Plants containing said trait exhibit the phenotype ofcross-incompatiblity. The present invention also relates to newcross-incompatible plants, including inbred, hybrid, haploid, apomicticand/or genetically engineered plants, containing the teosinte crossingbarrier trait. The cross-incompatible plants containing the teosintecrossing barrier trait described herein can be used in commercial fieldsto prevent the indiscriminate hybridization of these plants fromundesired lines, such as from genetically engineered plants.

BACKGROUND OF THE INVENTION

Maize (Zea mays L.), also known as corn, is a major worldwide crop thathas a number of practical uses. Maize is used as a food source for bothhumans and animals as well as a source of carbohydrates, oil, proteinand fiber. Many products are produced or extracted from maize, such ascorn syrup, adhesives, food thickeners, industrial and medicalabsorbants, ethanol, as well as many other products.

Maize can be bred by self-pollination or cross-pollination. Maize hasseparate male (called the tassel) and female (called the ear)inflorescences on the same plant. Maize is naturally pollinated whenwind blows pollen from the tassels to the silks that protrude from thetops of the developing ears.

Most maize is produced from hybrid seed. The production of hybrid maizeseed requires the elimination or inactivation of pollen produced by thefemale parent. Incomplete removal or inactivation of the pollen providesthe potential for self-pollination. This inadvertently self-pollinatedseed may be unintentionally harvested and packaged with hybrid seed.Several methods have been developed which can be used to control malefertility and thus prevent self-pollination. These methods includemanual or mechanical emasculation (commonly referred to as detasseling),cytoplasmic male sterility, genetic male sterility, gametocides and thelike.

Most frequently, hybrid maize seed is produced by manual or mechanicaldetasseling. This method works as follows. Typically, alternate stripsof two inbred varieties of maize are planted in a field and thepollen-bearing tassels are removed from the inbred which is to be usedas the female, prior to pollen being shed. As long as there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the male inbred, andthe resulting seed is a hybrid.

One of the problems with detasseling is that it is laborious (and henceexpensive) and is sometimes, unreliable. One alternative, to detasselinginvolves the use of cytoplasmic male sterile (CMS) inbreds. Cytoplasmicmale sterility is governed by factors in the maternal cytoplasm thatinduce sterility. Cytoplasmic male sterility can be used forreproduction of the female by fertilization with fertile pollen (i.e.,from a plant which is not cytoplasmically male sterile).

Another alternative to detasseling involves the use of genetic malesterility. Genetic male sterility is governed by nuclear factors thatinduce sterility and inhibit the normal development of the anthers andpollen. Genetic male sterility is inherited according to Mendelianprinciples in which the alleles for sterility are recessive (ms) to thealleles for pollen fertility (MS). There are many methods for conferringgenetic male sterility. For example, U.S. Pat. Nos. 4,654,465 and4,727,219 disclose the use of multiple mutant genes at separatelocations within the genome to confer male sterility. U.S. Pat. Nos.3,861,709 and 3,710,51 1 describe the use of chromosomal translocationsfor conferring genetic male sterility. Other methods involve deliveringinto a plant a gene encoding a cytotoxic substance associated with amale tissue specific promoter or an antisense system in which a genecritical to fertility is identified and an antisense to that geneinserted in the plant (see EP 329 308 and WO 90/08828).

Another alternative to detasseling is the application of certainchemicals referred to as gametocides, pollen suppressants or chemicalhybridizing agents. These chemicals block or kill viable pollenformation and hence produce a transitory male sterility. However, thereare a number of factors which affect the usefulness of these chemicalssuch as the expense of the chemicals, genotype specificity and the timeof the application.

Homozygous inbred maize lines are required from the production of maizehybrids. Homozygous inbred lines can be produced via pedigree breeding,where two maize inbred lines, each of which possesses different sets ofdesirable characteristics, are crossed. Superior plants are selected forthe traits of interest and then selfed for a number of generations inorder for the line to become increasingly inbred. This process ofcontinued selfing and selection is continued for five or moregenerations. The result of such breeding is the production of lineswhich are genetically homogenous or inbred. Typically, the developmentperiod for an inbred line using this method is a minimum of five years,if not longer.

One way for reaching homogeneity more quickly or completely whendeveloping an inbred line is through the use of haploid plants. Haploidsare plants which contain only one-half of the chromosome number presentin somatic cells, which are cells other than haploid cells. There are afew maize plants which are known to generate haploids spontaneously. Forexample, plants are known which possess an indeterminate gametophyte(ig) gene which generate haploids. Additionally, a line known as ‘Stock6’ (See, Birchler, J. A., “Practical Aspects of Haploid Production,” TheMaize Handbook, Freeling and Walbot (eds). pp. 386-388 (1996)) possessesa propensity to generate haploids. Utilization of either ig or ‘Stock 6’in a cross will result in the production of some haploid plants in theprogeny.

Alternatively, haploid plants can be produced using techniques known inthe art. One such technique is anther culture. Anther culture is amethod by which large numbers of haploids can be produced directly fromanthers in vitro. Generally, anther culture involves isolating immatureanthers,from plants and placing them onto a medium which induces thecells within the anther, which would normally be destined to becomepollen grains, to begin dividing and form a cell culture from which thehaploid plants can be regenerated. A number of techniques for carryingout anther culture are known in the art and are disclosed in J. M.Dunwell, “Anther and Ovary Culture”, In S W J Bright and M G K Jones,(eds.), Cereal Tissue and Cell Culture, Martinus Nijhoff Publisher,1985, Dordrecht, pp. 1-44 and U.S. Pat. Nos. 5,306,864, 5,322,7895,445,961, and 5,602,310. Another method which can be used to producehaploids is microspore culture. Microspore culture is similar to antherculture except that microspores are used instead of anthers to producehaploid plants (See, Coumans, et al., Plant Cell Reports 7:618-621(1989); Pescitelli et al., Plant Cell Reports, 7:673-676 (1989)). Theadvantage of anther or microspore culture is that it makes it possibleto test a larger number of new mutations and gene combinations and toselect among those for desirable traits.

Haploids obtained either spontaneously or from anther or microsporeculture, are sterile. To remedy this sterility, the chromosome number ofthe haploid can be doubled. Sometimes, the chromosome doubling can occurspontaneously. However, many times an agent must be used to effect thechromosome doubling. Agents which can be used to effect such chromosomedoubling, include, but are not limited to, colchicine or a mitoticspindle inhibitor. The doubling of the chromosome number results indoubled haploid plants which are completely fertile and inbred. Becausethese doubled haploid plants breed true, it makes the selection processfor desirable traits more efficient. Such doubled haploid plantspossessing desirable traits or characteristics can be used in pedigreebreeding to produce commercially valuable hybrids.

There exists a number of different methods for introducing a desiredtrait or characteristic into a targeted maize germplasm source, whethersuch maize germplasm source is an inbred or hybrid plant.

In a one method, backcrossing is used to introduce a desired trait orcharacteristic into a targeted maize germplasm source (called arecurrent parent) by crossing the recurrent parent with a donor plant(which is not the recurrent parent) which expresses certain traits ofinterest, such as, but not limited to, disease resistance, high yieldpotential, good stalk strength, reasonable drought tolerance, etc. Whilethe donor plant is preferably an inbred, it can also be any plantvariety which is cross-fertile with the recurrent parent. The progenyresulting from this crossing are then backcrossed to the recurrentparent, progeny possessing desirable traits identified, and the processrepeated. The process of backcrossing to the recurrent parent andselecting for the desired traits is repeated for five or moregenerations. The progeny resulting from this process are homozygous forloci controlling the trait(s) being transferred but will be like therecurrent parent. The last backcross generation is then selfed in orderto provide for pure breeding progeny for the gene(s) being transferred.

In a second method, apomixis can be used to introduce a desired trait orcharacteristic into a targeted maize germplasm source. Apomixis involvesthe production of hybrid seed without sexual reproduction. Apomixis is anatural method of reproduction in some plants and results in offspringthat are genetically identical (i.e. clones) to the mother plant, thusallowing improved hybrids, to breed true. This would permit commercialproducers and resource-poor farmers to replant seeds they produce, astrategy not practical with hybrid varieties available today for cropssuch as maize. U.S. Pat. No. 5,710,367, herein incorporated byreference, describes apomictic maize that have two unidentified geneswhich are believed to control apomictic development of the egg, as wellas certain methods for making apomictic maize.

In a third method, genetic engineering can be used to introduce adesired trait or characteristic into a targeted maize germplasm source.Genetic engineering refers to the sophisticated, artificial techniqueswhich are used to transfer genes from one organism to a recipientorganism. In agriculture, genetic engineering is used to create newplant varieties containing genes from other organisms which provide therecipient plant with improved traits, such as, but not limited to,improved yield, color, height, tolerance to frost, insect or diseaseresistance. However, there has been a great deal of apprehension aboutthe release of genetically engineered plants into the environment. Inthe United States, the U.S. Department of Agriculture, the Food and DrugAdministration and the Environmental Protection Agency, all regulate theuse of genetically engineered organisms, including geneticallyengineered plants.

One of the biggest concerns regarding genetically engineered orgenetically modified (hereinafter “GM”) crops involves the possiblecontamination of non-GM crops by GM crops. Specifically, there isconcern that transgenes contained in GM crops will travel, eitherthrough pollen or seed, to adjoining fields where non-GM crops are beingcultivated and pollinate the non-GM crops. This so-called “pollencontamination” or “indiscriminate hybridization” can be costly to afarmer engaged in farming using non-GM crops. Crop isolation distances,crop rotational and management practices have been developed in aneffort to alleviate the problem of “pollen contamination”. For example,in the U.S., it is recommended that an isolation distance of 203.1meters (about 0.126 miles) be established between GM and non-GM maizefields for the purposes of seed production. The presence of naturalbarriers can be used to reduce this isolation distance.

Thereupon, it is readily apparent that cross-pollinating plants, such asmaize, require a mechanism for preventing indiscriminate hybridization,especially from GM plants. Although differences in timing and isolationdistance may contribute to reproductive isolation, physiologicalbarriers often are sufficient to prevent crossing, especially amongwind-pollinated species. One such possible mechanism for preventing suchindiscriminate hybridization is cross-incompatibility. However, incontrast to self-incompatibility, cross-incompatibility (hereinafterreferred to as “CI”) is poorly characterized both genetically andphysiologically.

In domesticated maize, CI ranges in degree from creating a preferenceamong pollen classes up to preventing seed set. Genes responsible forthese effects are called gametophyte factors (hereinafter “Ga”) becausethe efficiency of pollen function is affected (see Nelson, O. E., TheMaize Handbook, M. Freeling and V. Walbot, eds. Springer-Verlag (1993)).Ga factors conferring only a preference among pollen genotypes arecryptic, influencing the transmission of linked genes and thecompetitive ability of pollen in mixtures. Examples that involverecognition between corresponding alleles in pollen and silks are Ga2,Ga4, Ga8, and certain combinations involving Ga1. Incompatibilityleading to failure of seed set occurs in conjunction with the strongallele of Ga1, specifically when Ga1-s Gal-s plants are pollinated withga1 gal, the cross used to isolate commercial popcorns from the pollenof other maize plants. As a system of isolation, Ga1-s is imperfectbecause some maize strains carry a gal-s or yet another allele, Gal-m,which permits them to cross to strains containing both ga1 and Ga1-s. Inthese strains, the barrier breaks down.

Thereupon, there is a need in the art for genetically andphysiologically well-characterized cross-incompatibility systems inmaize which prevent the indiscriminate hybridization of maize plantsfrom unwanted pollen sources.

SUMMARY OF THE INVENTION

The present invention relates to a cross-incompatible maize plantcontaining a teosinte crossing barrier (“TCB”) trait. Across-incompatible maize plant containing the TCB trait plant (1) failsto set seed when pollinated by plants lacking the TCB trait but setsseed when pollinated by plants carrying the TCB trait; and/or (2)maintains functional pollen and sets seed when pollinated by itself orcauses other maize plants to set seed when pollinated by said plant. Inaddition to the TCB trait, the cross-incompatible maize plants of thepresent invention further comprise a gene cluster within its genomewherein said gene cluster is located on the short arm of chromosome 4between map units 40-85. The gene cluster of the present inventioncomprises (1) a Tcb locus within its genome; and/or (2) at least onemodifier gene within its genome. The Tcb locus is located on the shortarm of chromosome 4 about 6 map units distal to the sugaryl gene andbout 40 map units from the Ga1gene. The cross-incompatible maize plantsof the present invention can be inbred, hybrid, haploid, apomicticand/or genetically engineered plants.

The present invention further relates to a cross-incompatible maizeplant exhibiting a TCB trait wherein said TCB trait is derived fromW22-TCB. Such a cross-incompatible maize plant can further contain agene cluster within its genome wherein said gene cluster is located onthe short arm of chromosome 4 between map units 40-85. The gene clustercan contain (1) a Tcb locus within its genome; and/or (2) at least onemodifier within its genome. The cross-incompatible maize plant of thepresent invention can be an inbred, hybrid, haploid, apomictic and/orand genetically engineered plant.

The present invention further relates to a process for obtaining a firstinbred maize plant, which when crossed with a second inbred maize plant,produces hybrid maize seed, which when planted and grown under plantgrowth conditions, produces hybrid maize plants which exhibit the TCBtrait of cross-incompatibility. The first step of the process involvesselecting a first donor parental maize plant from a population of maizeplants which are cross-incompatible and contain a TCB trait. The secondstep involves crossing the selected donor first parental maize plantwith a second parental maize plant. The second parental maize plantpreferably contains genes which encode for desirable traits in hybridcombination. In addition, the second parental maize plant may or may notbe cross-incompatible and contain the TCB trait. The third step involvescollecting the resulting maize seed. The fourth step involves plantingand growing the resulting seed under plant growth conditions. The fifthstep involves screening the resulting plant population for the presenceof the TCB trait identified in the first step. The final step involvesselecting plants from said population containing the TCB trait forcross-incompatibility for further crossings and screenings until a maizeline is obtained which is homozygous for the TCB trait so as to providethis trait in an inbred to be used in hybrid combination.Cross-incompatible inbred maize plants containing the TCB trait producedas a result of this process are also encompassed by the presentinvention.

The present invention further relates to a process of producing across-incompatible hybrid maize plant containing a TCB trait. The firststep of the process involves crossing a cross-incompatible inbred maizeplant containing the TCB trait for cross-incompatibility with a secondmaize inbred line to produce a segregating plant population. Preferably,the second maize inbred line contains genes encoding desirablephenotypic traits. In addition, the second maize inbred line may or maynot be cross-incompatible and contain the TCB trait. The second step ofthe process involves collecting the resulting hybrid maize seed. Thepresent invention also relates to a cross-incompatible hybrid maizeplant containing a TCB trait produced as a result of this process.

The present invention further relates to a process for selecting a firstdonor parental maize plant suitable for use in producing an inbred maizeplant, which inbred maize plant, if crossed with a second inbred maizeplant, produces a hybrid maize plant which is cross-incompatible andcontains a TCB trait. The method involves the step of analyzing eachplant from a population of maize plants for plants containing a TCBtrait. The method can further involves the steps of analyzing the DNA ofeach plant from the population of maize plants for a gene cluster withineach plant's genome wherein said gene cluster is located on the shortarm of chromosome 4 between map units 40-85. Additionally, the DNA ofeach plant from the population of maize plants can be further analyzedfor: (1) a Tcb locus located on the short arm of chromosome 4 about 6map units distal to the sugary1 gene and about 40 map units from thegene Ga1gene; and/or (2) at least one modifier gene. The presentinvention also relates to a cross-incompatible first donor parentalmaize plant containing a TCB trait produced as a result of this process.

The present invention further relates to a process for selecting across-incompatible hybrid maize plant containing a TCB trait. The methodinvolves the step of analyzing each plant from a population of hybridmaize plants for the TCB trait. The method can further involves thesteps of analyzing the DNA of each plant from the population of maizeplants for a gene cluster within each plant's genome wherein said genecluster is located on the short arm of chromosome 4 between map units40-85. Additionally, the DNA of each plant from the population of maizeplants can be further analyzed for: (1) a Tcb locus located on the shortarm of chromosome 4 about 6 map units distal to the sugary1 gene andabout 40 map units from the gene Ga1gene; and/or (2) at least onemodifier gene. The present invention also relates to across-incompatible hybrid maize plant containing a TCB trait produced asa result of this process.

The present invention further relates to a process of controllinghybridization of a maize plant in a field and/or a phytotron orgreenhouse. The process involves the step of planting in a field and/orphytotron or greenhouse a cross-incompatible maize plant containing thepreviously described TCB trait.

The present invention further relates to a process of controllinghybridization of inbred maize plants in a field being used in hybridseed production. The process involves the step of planting in a fieldand/or a phytotron or greenhouse being used for hybrid seed production,a cross-incompatible inbred maize plant containing the previouslydescribed TCB trait.

Other objects, advantages and features of the present invention willbecome apparent from the following specification when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a chromosome (linkage) map showing the location of Tcbrelative to flanking genes on of the short arm of chromosome 4 in Zeamays.

FIG. 2A-2F shows ears from crosses designed to distinguish betweenteosinte-derived incompatibility genes segregating in W22 backcrossprogeny. Pollen from a colorless kernel, true breeding TCB strain wasmixed with ga1 tcb1 pollen that confers kernel color and then placed onsilks of plants in the test strain (See FIGS. 2A and 2D). Pollen fromeach of the test plants was used in crosses to Gal-s (FIGS. 2B and 2E)and Tcb1 (FIGS. 2C and 2F) silks. Two classes of plants were present.The first class of plants are shown in FIG. 2A-FIG. 2C whose silks didnot discriminate against the colored tracer pollen and whose pollen wasunreceptive on both testers. These plants lack both Ga1and Tcb1. Thesecond class of plants were those plants shown FIG. 2D-FIG. 2F whosesilks were unreceptive to the tracer pollen and whose own pollen waspoorly receptive on the Ga1-s tester but receptive on Tcb1. These plantslacked Gal but carried Tcb1.

FIG. 3A shows the F1 heterozygous genotype giving parental andrecombinant classes I, II and III used to located tcb. FIG. 3B shows achromosome 4 genetic map indicating location of tcb1 relative to visualand molecular markers in the proximal region of the short arm. Thecentromere is indicated by a filled circle.

FIG. 4 shows the difference between the teosinte incompatibility (“TIC”)phenotype and the TCB trait of the present invention.

FIG. 5A-5B shows shows Tcb1 and Gal pollen competition. FIG. 5A showsthe success of gal tcb1 pollen (colored kernel strain) in competitionwith gal Tcb1 (colorless kernel strain). Pollen from the two sources wasmixed then placed on silks of female strains all of which confercolorless kernels. FIG. 5B shows the success of ga1 tcb1 pollen (coloredkernel strain) in competition with Gal-s tcb1 (colorless kernel strain).Pollen from the two sources was mixed then placed on silks of the sameset of female strains, all of which confer colorless kernels.

INTRODUCTION

The present invention relates to a teosinte crossing barrier (“TCB”)trait. Plants containing this trait exhibit the phenotype ofcross-incompatiblity. The present invention also relates to newcross-incompatible plants, including inbred, hybrid, haploid, apomicticand genetically engineered plants, containing the teosinte crossingbarrier trait. The cross-incompatible plants containing the teosintecrossing barrier trait described herein can be used in commercial fieldsto prevent the indiscriminate hybridization of these plants fromundesired lines, such as from genetically engineered plants.

DEFINITIONS

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

As used herein, the term “allele(s)” means any of one or morealternative forms of a gene, all of which alleles relate to one trait orcharacteristic. In a diploid cell or organism, the two alleles of agiven gene occupy corresponding loci on a pair of homologouschromosomes.

As used herein, the term “corn” means any variety, cultivar orpopulation of Zea mays L.

As used herein, the term “cross-incompatibility” refers to the inabilityof two strains or species to hybridize and/or cross-pollinate.

As used herein, the term “elite” means a plant or variety possessingcertain favorable traits, such as, but not limited to, high yield, goodgrain quality and disease resistance.

As used herein, the term “expressed sequence tag” or “EST” means partialgene sequence data of a cDNA clone which provides a sequence tag for agene.

As used herein, the term “gene cluster” refers to an identifiedchromosomal region containing genetic material (such, as, but notlimited to one or more genes or alleles) which expresses a desiredtrait.

As used herein, the term “heterozygous” means a genetic conditionexisting when different alleles reside at corresponding loci onhomologous chromosomes.

As used herein, the term “homozygous” means a genetic condition existingwhen identical alleles reside at corresponding loci on homologouschromosomes.

As used herein, the term “hybrid” means any offspring of a cross betweentwo genetically unlike individuals (Rieger, R., A Michaelis and M. M.Green, 1968, A Glossary of Genetics and Cytogenetics, Springer-Verlag,N.Y.).

As used herein, the terms “hybridize” or “hybridization” refers to theformation of offspring between different or unlike parents.

As used herein, the term “inbred” means a substantially homozygousindividual or variety.

As used herein, the term “introgressed” means the entry or introductionof a gene or gene cluster from one plant into another. As used herein,the term “introgressing” means entering or introducing a gene or genecluster from one plant into another.

As used herein, the term “isozyme” means detectable variants of anenzyme, the variants catalyzing the same reaction(s) but differing fromeach other, for example, in primary structure and/or electrophoreticmobility.

As used herein, the term “locus” or “loci” means the site in a linkagemap or on a chromosome where the gene for a particular trait is located.Any one of the alleles of a gene may be present at this site.

As used herein, the term “maize” means any variety, cultivar orpopulation of Zea mays L.

As used herein, the term “map unit” means a unit of distance in achromosome map (linkage map), frequently measured in centiMorgans.Specifically, a mapping unit is the distance between two linked genepairs where 1 percent of the products of meiosis are recombinant.

As used herein, the term, “microsatellite” or “simple sequence repeat(SSR) or “dinucleotide repeat” or “trinucleotide repeat” or“tetranucleotide repeat” all refer to stretches of DNA consisting oftandemly repeating di-, tri-, tetra-, or penta-nucleotide units. An SSRregion can be as short as two repeating units, but more frequently is inexcess of 8-10 repeating units. Simple sequence repeats are common invirtually all eukaryotic genomes studied and have been identified asuseful tools for the study of genetic polymorphisms.

As used herein, the term “modifier gene” refers to a gene that affectsthe phenotypic expression of another gene.

As used herein, the term “molecular marker”, includes, but is notlimited to, an amplified fragment length polymorphism (AFLP), anexpressed sequence tag (EST), a restriction fragment lengthpolymorphism, (RFLP), a microsatellite (also known as a “simple sequencerepeat” (SSR)),a single nucleotide polymorphism (SNP), or an isozymemarker which defines a specific genetic and chromosomal location.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which maize plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants, or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, seeds, husks, stalks, roots, root tips,anthers, silk and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants sharing a common genetic derivation.

As used herein, the term “Restriction Fragment Length Polymorphism” or“RFLP” means a variation between individuals in DNA fragment sizes cutby specific restriction enzymes. Polymorphic sequences which result inRFLPs are used as markers on both physical maps and genetic linkagemaps.

As used herein, the term “Single Nucleotide Polymorphism” or “SNP”refers to a point mutation that occurs in greater than 1% of thepopulation.

As used herein, the term “variety” or “cultivar” means a group ofsimilar plants that by structural features and performance can beidentified from other varieties within the same species.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a teosinte crossing barrier trait (“TCBtrait”) for cross-incompatibility. Cross-incompatible maize plants whichcontain the TCB trait (1) fail to set seed when pollinated with pollenfrom maize plants lacking the TCB trait but sets seed when pollinated byplants carrying the TCB trait (see rows 1-120 in Example 2) and/or (2)maintain functional pollen and set seed when pollinated by itself orcauses other maize plants to set seed when pollinated by said plant. TheTCB trait of the present invention can be introgressed into maize plants(such as inbred, hybrid, haploid, apomictic and/or geneticallyengineered plants) which do not contain this trait in order to producenew cross-incompatible maize plants containing the TCB trait (SeeExample 2).

The TCB trait of the present invention is comprised of a gene clusterwhich is expressed dominantly and located on the short arm (frequentlyreferred to as “S”) of chromosome 4, between map units 40-85 (shown inFIG. 1). Preferably, the gene cluster is located between map units 40-80on the short arm of chromosome 4 (shown in FIG. 1 and FIG. 3B). Mostpreferably, the gene cluster is located between map units 66-75 on theshort arm of chromosome 4 (See FIG. 3B).

The gene cluster preferably contains a locus which resides within thechromosomal position discussed previously and is referred to herein asthe “teosinte crossing barrier” or “Tcb” locus. The Tcb locus is locatedat about 6 map units (or centiMorgans) distal to the sugary1 marker onchromosome 4S, about 40 map units (or centiMorgans) from the Ga1 marker.The Tcb locus contains genes which govern the compatibility of pollenand silks. One property encoded by at least one gene(s) comprising theTcb locus is referred to herein as the “silk effect” function. Morespecifically, cross-incompatible maize plants containing the Tcb (whichis either homozygous or heterozygous) and which exhibit the silk effect,when used as a female parent, fail to set seed when pollinated withpollen from maize plants which do not exhibit the TCB trait forcross-incompatibility, such as, but not limited to dent (ga1) and pop(Ga1-s) lines. Maize plants heterozygous for the Tcb locus and whichexhibit the silk function, reject both ga1 and Ga1-s pollen at a levelsimilar to the rejection of ga1 pollen by homozygous Ga1-s silks.However, while cross-incompatible maize plants containing the Tcb locusand which exhibit the silk effect, when used as a female parent, fail toset seed when pollinated with pollen from other maize plants which donot contain the Tcb locus and which do not exhibit the silk effect, suchas dent (ga1) or pop (Ga1-s) corn lines, cross-incompatible maize plantscontaining the Tcb locus and which exhibit the silk effect, when used asa female parent, accept pollen and set seed when pollinated by maizeplants containing the Tcb locus exhibiting the silk effect. The Tcbproperties of a barrier to crossing, as a female parent, and the abilityto overcome the barrier, as a pollen parent, are inherited together andmay be functions of the same or different genes of the Tcb locus.

A second property encoded by at least one gene(s) comprising the Tcblocus is referred to herein as the “pollen effect”. More specifically,maize plants containing the Tcb locus and which exhibit the polleneffect, when used as a male parent, maintain functional pollen and canfertilize themselves, as well as certain other maize plants, such asdent (gal) lines. Cross-incompatible maize plants containing the Tcblocus, and which exhibit the silk and pollen effect (a) reject pollenfrom dent (gal) lines when used as a female parent; and (b) maintainfunctional pollen and can pollinate themselves as well as dent (ga1)lines, when used as a male parent.

While it is preferred herein that cross-incompatible maize plantscontaining the TCB trait and the Tcb locus possess at least one gene(s)encoding the silk and pollen effect functions, maize plants containingthe TCB trait and the Tcb locus and possessing at least one gene(s)encoding only the silk effect or pollen effect function are alsoencompassed by the present invention.

In addition to the Tcb locus, the gene cluster described herein can alsocontain at least one modifier gene which modifies the effect of the Tcblocus. Presently, the inventors believe that at least one modifier genewhich modifies the effect of the Tcb locus is located about 6.5 mapunits or centiMorgans from the Tcb locus in the direction of the Gal1marker (see FIG. 1). Other modifier genes which modify the effect of theTcb locus and which are located (1) within the gene cluster; (2) outsidethe gene cluster on chromosome 4; and/or (3) on one or more chromosomesother than chromosome 4, are also contemplated herein. Moreover, thegene cluster described herein may also contain other genes and suchgenes are also contemplated to be within the scope of the presentinvention.

The prior art, specifically, Kermicle, J. L., et al., in Maydica35:399-408 (1990) is distinguishable from the present invention in thatsaid prior art discuses the phenotype referred to as teosinteincompatibility or “TIC”. The TIC phenotype is not the same as the TCBtrait. As shown in FIG. 4, the TIC phenotype discussed in Kermicle etal. is described as including TIC-CP1 (Gal-m) (which is associated withthe pollen effect function Tcb), TIC-CP2 as well as some undefinedfunctions. TIC-CP2 arose as a rare variant from TIC that lost the femalefunction of pollen rejection. Maize plants containing TIC-CP2, when usedas a male parent, were capable of pollinating maize plants containingTIC and dent (gal) but not pop (Gal-s) lines. In contrast, the presentinvention relates to the TCB trait. The TCB trait of the presentinvention includes the Tcb locus and one or more modifier genes whichmodify the effect of the Tcb locus. The Tcb locus of the presentinvention contains at least one gene(s) encoding the silk effect and/orpollen effect functions. The undefined functions generally referred toin Kermicle et al. have been discovered by the present inventors torelate to the TCB trait and comprise of one or more modifier genes andat least one gene(s) encoding the silk effect function.

An example of a maize stock which contains the TCB trait ofcross-incompatiblity is dent inbred W22-TCB. Cross-incompatible inbredW22-TCB contains the gene cluster described herein. More specifically,the gene cluster contains the Tcb locus and at least one gene(s) whichencode for the silk and pollen effect functions and at least onemodifier gene to the Tcb locus. In addition, W22-TCB also containsTIC-CP1. Two thousand five hundred (2500) seeds of inbred line W22-TCBhave been placed on deposit with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va., 20110-2209 under DepositAccession Number PTA-1601 on March 30, 2000. This deposit was made incompliance with the Budapest Treaty requirements that the duration ofthe deposit should be for thirty (30) years from the date of the depositor for five (5) years after the last request for the deposit at thedepository or for the enforceable life of a U.S. patent that maturesfrom this application, whichever is longer. These seeds will bereplenished should it become non-viable at the depository.

The present invention further contemplates the use of the TCB trait fromdent inbred W22-TCB in crosses to develop new inbred and hybrid plantscontaining the TCB trait of cross-incompatibility and commerciallydesirable characteristics. Such new inbred and hybrid plants can bedeveloped using plant breeding and molecular biology techniques known inart. For example, inbred W22-TCB can be used to create a first inbreddent line which is cross-incompatible and contains the TCB trait. Thisfirst inbred dent line containing the TCB trait can be crossed with asecond inbred dent line to create a cross-incompatible dent hybridhaving commercially desirable traits. Additionally, W22-TCB and inbredsderived therefrom can be used in anther tissue culture to createhaploids and doubled haploid plants as well as apomictic maize plants.Moreover, the present invention further contemplates new maizecross-incompatible inbred, hybrid, haploid, apomictic and/or andgenetically engineered plants containing the TCB trait wherein the TCBtrait is derived from W22-TCB.

Molecular biology techniques can be used to identify plants whichcontain the TCB trait. For example, molecular markers can be used toidentify the gene cluster described herein. Any molecular markers whichcan be used to identify the gene cluster associated with the TCB traitare contemplated as being within the scope of the present invention. Forexample, the markers including and between phi021 and nc005 shown inFIG. 1 and the markers including and between umc 1117 and bnlg 490,particularly, mmc0471 shown in FIG. 3B, can be used to identify the genecluster. Thereupon, between deposited dent inbred W22-TCB and themolecular markers described herein, other sources of genetic materialcontaining the TCB trait and gene cluster described herein can and willof course be located as the present invention now allows this materialto be identified using routine techniques known in the art. Morespecifically, the TCB trait and gene cluster described herein can beused to identify said TCB trait or an analogous trait and analogousgenes, gene clusters and/or molecular markers in other cross-pollinatingmonocotyledonous and dicotyledonous plants, such as, but not limited torapeseed and rye, using techniques known in the art. The identificationof homologous cross-incompatibility systems which function in a mannersimilar to the cross-incompatibility system described herein in maizeare particularly important in rapeseed. Rapeseed is a cross-pollinatingspecies (rapeseed contains a self-incompatibility system which preventsit from self-pollinating with itself). Unfortunately, reports ofcontamination of non-GM rapeseed varieties with transgenes from GMrapeseed varieties is known in the art. The development ofcross-incompatible rapeseed which function similar to thecross-incompatible maize plants described herein would be beneficial toprevent the undesired and indiscriminate hybridization of rapeseed by GMrapeseed varieties.

The TCB trait described herein can be introgressed into maize plantsusing plant breeding techniques known in the art. Specifically, maizeplants exhibiting the TCB trait herein, can be crossed as the pollenparent with other donor maize plants containing genes which encode forcommercially desirable traits to create cross-incompatible inbred,hybrid, haploid, apomictic and/or and genetically engineered maizeplants containing the previously described TCB trait. Cross-incompatibleinbred maize lines containing the TCB trait within their genome whencrossed with a second inbred maize line, can be used to produce a hybridmaize plant which is cross-incompatible and contains the TCB trait. Suchcross-incompatible inbred maize plants can be developed by selecting afirst donor parental maize plant from a population of maize plants whichis cross-incompatible and contains the TCB trait. When a suitable donorparental maize plant containing the TCB trait is identified, it iscrossed with a second donor parental maize plant. The second donorparental maize plant may or may not be cross-incompatible and containthe TCB trait. In addition, the second donor parental maize plantpreferably containing genes which encode for commercially desirabletraits in hybrid combination. Such commercially desirable traitsinclude, but are not limited to, insect resistance (such as, but notlimited to, resistance to mites, aphids, beetles, spiders, etc),nematode resistance (such as, but not limited to, resistance to FallArmyworm, Western Rootworm, etc.), resistance to disease (such as, butnot limited to, resistance to Northern Leaf Blight, Southern LeafBlight, Southern Rust, Stewart's Wilt, Corn Lethal Necrosis, Head Smut,Maize Chlorotic Dwarf Virus, Maize Chlorotic Mottle Virus, Maize DwarfMosaic Virus, etc.), etc. The resulting seeds are collected, planted andgrown under plant growth conditions. The resulting plant population isthen screened for those plants which contain the previously describedTCB trait. These plants are then selected for further crossings underself-pollinating or sib-pollinating conditions until an inbred line isobtained which is homozygous for the TCB trait. This cross-incompatibleinbred maize line can then be used in selfing, backcrossing, hybridproduction, crosses to populations, and the like.

For example, an inbred maize line containing commercially desirablecharacteristics (the recurrent parent) can be crossed to a donor inbred(the non-recurrent parent) that carries the TCB trait described hereinwithin its genome. The progeny of this cross is then mated back to therecurrent parent. Selections are made from the resulting progeny forthose plants containing the TCB trait of cross-incompatability to betransferred from the non-recurrent parent. After three, preferably four,or more preferably five or more generations of backcrosses with therecurrent parent with selection for cross-incompatability, the progenywill be heterozygous for loci controlling the TCB trait ofcross-incompability, but will be like the recurrent parent for most oralmost all other genes.

Various laboratory-based techniques, such as marker-assisted selectionand other techniques known in the art, can be used in backcrosses toidentify the progenies having the highest degree of genetic identitywith the recurrent parent. This allows for the production andidentification of inbred maize lines having at least 90%, preferably95%, and more preferably at least 99% genetic identity with therecurrent parent.

The cross-incompatible inbred maize lines described herein can be usedin additional crossings to create cross-incompatible hybrid plants. Forexample, a first cross-incompatible inbred maize plant containing theTCB trait in its genome can be crossed as the pollen parent with asecond inbred maize line. Preferably, the second inbred maize linepossesses commercially desirable traits such as, but not limited to,insect resistance (such as, but not limited to, resistance to mites,aphids, beetles, spiders, etc), nematode resistance (such as, but notlimited to, resistance to Fall Armyworm, Western Rootworm, etc.),resistance to disease (such as, but not limited to, resistance toNorthern Leaf Blight, Southern Leaf Blight, Southern Rust, Stewart'sWilt, Corn Lethal Necrosis, Head Smut, Maize Chlorotic Dwarf Virus,Maize Chlorotic Mottle Virus, Maize Dwarf Mosaic Virus, etc.), etc. Inaddition, this second inbred maize line may or may not contain the TCBtrait for cross-incompatibility. The resulting seed is then collected,planted and grown under plant growth conditions. The resulting plantpopulation is then screened for hybrid plants containing the TCB traitfor cross-incompatibility. These cross-incompatible hybrid plantsexhibiting the TCB trait are then selected.

The cross-incompatible maize plants containing the TCB trait describedherein can be used to control hybridization in a field and/or phytotronor greenhouse for the purpose of developing new inbred or a hybrid maizeplants. Specifically, the cross-incompatible maize plants of the presentinvention can be planted in a field and/or phytotron or greenhouse andused to create new inbred and hybrid maize plants having certaincommercially desirable traits without concern that these maize plantswill be indiscriminately hybridized and contaminated by pollen fromunwanted maize sources (such as GM maize plants) located either in thesame field or in a nearby field or in the same phytotron or greenhouse.For example, cross-incompatible inbred maize plants containing withintheir genome the TCB trait described herein can be planted in a fieldand/or phytotron or greenhouse being used for hybrid seed production.These cross-incompatible inbred maize plants may be planted in or nextto a field or in the same phytotron or greenhouse containing GM maizeplants. These cross-incompatible maize plants can then be crossed toproduce hybrid seed. Because the inbred maize plants arecross-incompatible, they will not be fertilized by any pollenoriginating from the GM maize plants. Instead, these plants will only befertilized by pollen originating from a cross-incompatible maize plantcontaining the TCB trait described herein.

By way of example, but not limitation, examples of the present inventionshall now be given.

EXAMPLE 1 Identification and Characterization of the Teosinte CrossingBarrier Locus EXAMPLE 1a Materials and Methods

Table 1 below lists the incompatibility stocks used and gives theircompatibility relations. TABLE 1 Incompatibility Stocks Source of StockIncompatibility Genes Distinguishing Features ga1 tcb1 dent maize inbredW22 Receptive to all pollen; unable to fertilize Ga1-s/Ga1-s or TCB/−Gal-s tcb1 White Cloud Popcorn Homozygotes unreceptive hybrid andheterozygotes variably receptive to ga1 pollen; unable to fertilizeTCB/− Ga1-m tcb1 Central Plateau teosinte Receptive to all pollen; able48703 to fertilize Gal-s/Gal-s but not TCB/− TCB Central Plateauteosinte TCB/− unreceptive to tcb1; haplotype 48703 fertilizes allpistil genotypes. TCB/TCB weakly self-incompatible. ga1 Tcb1 CentralPlateau teosinte Tcb1/− unreceptive or partly 48703 receptive to tcb1depending on modifier constitution; unable to fertilize Gal-s/Gal-s

In order to standardize the genetic background, the genes of interestwere incorporated through backcrossing into the Midwestern US dentinbred W22, which lacks known incompatibility factors (ga1 tcb1). TheGa1-s counterpart line was developed by first crossing W22 with WhiteCloud hybrid popcorn, then backcrossing Ga1-s containing progeny to W22for five generations before self-pollinating to establish a homozygouslineage. Incompatibility genes from Central Plateau teosinte collection48703 (Wilkes, “Teosinte: the closest relative of maize”. The BusseyInstitute of Harvard University, Cambridge, Mass. (1967)) weretransferred to maize first by crossing to various ga1 tcb1 stocks, asavailable, for five generations and then successively to W22. Selectionfor strong incompatability resulted in the multifactoral haplotypedesignated TCB, established for routine use as a Tcb1 tester after threegenerations of crossing to W22. Ga1-m tcb1 and ga1 Tcb1 were isolatedfrom TIC, after additional generations of backcrossing (identified asclasses B and C, respectively, in Table 3 of Kermicle et al., Maydica35:399-408 (1990)). The experiment involving pollination first with ga1tcb1 and then a day later with the plant's own pollen was conductedusing stocks derived before incorporating TCB into inbred W22background.

EXAMPLE 1b Tcb1 Mapping

Crossing a Tassel seed5 (Ts5) strain of Tcb1 to the chromosome 4 testerstock virescent17 (v17), brown midrib3 (bm3), sugary1 (su1) (MaizeGenetics Cooperation Stock Center) and the F1 back to the recessivetester generated a 5-point testcross population for locating Tcb1relative to the four visual markers. Only the non-sugary kernel classwas characterized due to reduced viability and difficulty in classifyingthe virescent seedling phenotype within the sugary class. A sample ofnon-sugary kernels was field seeded, classified for v17, bm3 and ts5phenotypes to identify crossover classes, then crossed reciprocally withTCB/su1 to determine Tcb1:tcb1 composition. Additional v17-Su crossoverindividuals were identified as virescent seedlings in a greenhouseplanting, and then field transplants were classified for adult plantphenotypes and evaluated for Tcb1:tcb1. Five of the v17-Su1 crossoverswere established from a progeny in which virescent expression wasincompletely penetrant, hence the effective population size could not bedetermined. In the remaining three progenies studied, fifteen v17-Su1crossovers were present among 237 plants, or 6.3±1.6%, which compareswith 7.6±1.3% reported previously (Stinard, Maize Gen. Coop. Newsletter72:79 (1998)).

EXAMPLE 1c Simple Sequence Repeat Analysis

Simple Sequence Repeat (SSR) markers were chosen that were known orsuspected to map on the short arm of chromosome 4 based on data inMaizeDB. DNA was extracted from samples using the protocol described inDellaporta in “Plant DNA miniprep and microprep: versions 2.1-2.3,” TheMaize Handbook, Freeling and Walbot (eds.) pp 522-525 (1994). PCRreactions were performed on a PTC-200 Thermal Cycler (MJ Research). Theamplification conditions were the same as the “touchdown” profile ofSenior et al., Crop Sci, 38:1088-1098 (1998) except that the last cyclewas repeated 30 times instead of 20 times prior to terminating with acontinuous 4° C. cycle. The 15 μL reaction mix consisted of 3 pmoles ofeach primer, 2.5 mM MgCl₂, 100 μM each dNTP, 10 mM Tris pH 9, 50 mM KCl,0.1% Triton X-100, 1 mg/mL purified BSA (New England Biolabs), 0.6 unitsTaq DNA polymerase (Promega), and approximately 30 ng template DNA.After amplification, three μL of loading dye (30% glycerol., 0.25%bromophenol blue, 0.25% xylene cyanol) was added to each sample, and sixμM of each mix was electrophoresed on 4% Metaphor (FMC Bioproducts)agarose gels in 1×TBE (Sambrook et al., Molecular cloning. A LaboratoryManual. Cold Spring Harbor Lab Press, New York 1989). Afterelectrophoresis, gels were stained in 0.5 μg/mL ethidium bromide andvisualized on an UV transilluminator. Allelic constitution was firstdetermined for the v17 bm3 su1 multiple tester and the Ts5 Tcb1 stockfor all of the SSR markers. Those with detectable polymorphisms betweenthe two parental types were then tested on the recombinants between Ts5and su1.

EXAMPLE 1d Pollen Mixtures

In tests for preferential usage, approximately equal quantities of ga1tcb pollen and that of either Gal-s tcb1 or ga1 Tcb1 were thoroughlymixed and distributed to silks of various genotypes. The ga1 tcb sourceused confers colored kernels (R-sc plus other complementary genesrequired for aleurone color) whereas the remaining strains confercolorless kernels (r-g plus complementary color genes). For each mix,the proportion of viable pollen of the two classes was determined fromthe proportion of colored to colorless kernels obtained in crosses tocolorless ga1 tcb (two crosses per mix). Values ranged from 28.8 to54.2% colored. To standardize results across mixes, the proportion ofcolored kernels was divided by that determined for the two colorless ga1tcb females, then averaged across the eight mixes made for each type.This transformation expresses results for the test females relative toga1 tcb. Thus, if there is no difference in preference among pollenclasses the expected value is one, or 100%. Ears having fewer than 50kernels total were excluded from the calculation. Of the 144 crossesattempted in this experiment, 16 failed as anticipated because neitherclass of pollen was compatible with the pistil genotype, and four wereunsuccessful for reasons extraneous to compatibility. Percentage seedset was estimated as described in Kermicle et al., Maydica 35:399-408(1990).

EXAMPLE 1e Results

Three features of teosinte-derived incompatibility by Tcb1 were followedin a segregating testcross population. As female parent, plantscontaining Tcb1 discriminate against Tcb1-pollen (see FIG. 2). As maleparent, Tcb1 pollen not only functions preferentially on Tcb1-containingsilks but also is discriminated against on tcb1/tcb1 silks. Table 2below presents data testing inheritance of these features relative toone another and in relation to four visual marker loci located in theproximal region of chromosome arm 4S. This region was chosen as likelyto be of interest because only three percent sugary-1 kernels had beenrecovered in F2 progenies of TIC Su1/+su1 heterozygotes (Kermicle etal., Maydica 35:399-408 (1990)). TABLE 2 Location on maize chromosomearm 4S of Teosinte crossing barrier1 (Tcb1) relative to four visualmarkers, based on a testcross population produced by crossing Ts5 V17Bm3 Su1 (Tcb1)/ts5 v17 bm3 su1 (tcb1) heterozygotes to ts5 v17 bm3 su1females. Recombinant progeny grown from nonsugary kernels were evaluatedfor Tcb1 through reciprocal crosses with TCB Su1/+su1 and by crossing tohomozygous su1 % sugary % sugary % seed set kernels with kernels in withrecombinant crosses to Recombinant No. recombinant class as su1/su1females Tcb1:tcb1 class plants class as male female (no. of plants)constitution I. ts5 - V 17 6 70.8 3.8-21.9 54.5 (4) Tcb1 II. v17 - Bm3Group A 4 66.3 4.2-19.2 57.8 (3) Tcb1 Group B 5   0.0^(a) 28.1-35.3 50.3 (4) tcb1 III. bm3 - Su1 11   0.0^(b) 24.9-34.0  50.5 (7) tcb1^(a)zero kernels^(b)a total of 8 kernels

The backcross data are summarized according to single crossover classes,no multiple crossovers having been detected. A sample of six plantsrecombinant for ts5 and v17 (see region I of FIG. 3A) retained all threeTcb1 features. Conversely, eleven bm3-Su1 crossovers (region III)retained none. The nine v17-Bm3 crossovers (region II) fell into twoclasses: four retained all Tcb1 features whereas five retained none. Theregular cosegregation of the three features defines the teosintecrossing barrier1 locus. Being approximately midway between v17 and bm3,it maps to position 74 (see FIG. 3B) on the 1995 maize genetic map ofNeuffer et al., Mutants of Maize, Cold Spring Harbor Lab Press, New York(1997).

Simple Sequence Repeat (SSR) markers polymorphic between the multipletester and the Ts5 Tcb1 parent were tested retrospectively on therecombinants in the four regions between Ts5 and Su1 defined by thevisual markers and tcb1 (See FIG. 3B). This data places umc1117 andphi074 distal to Tcb1 between v17 and Ts5, bnlg490 proximal to Tcb1halfway between Tcb1 and bm3, and nc005 and bnlg1937 between bm3 andSu1. In this population mmc0471 was not separated from Tcb1.

Exclusion of tcb1 pollen by Tcb1/tcb1 plants in the foregoing linkagestudy was variable. Nine backcross plants of the Ts5 Tcb1 parental classproduced from 1.5 to 15.3% sugary kernels rather than the Mendelianexpectation of 25%. Similarly, the six ts5-V17 crossover plants rangedfrom 3.8 to 21.9% sugary. This compares with a range of only 1.1 to 3.8%in the reciprocal cross, for example, when pollen of the crossoverplants was put onto TCB Su1/+su1 females. This lower value is in thesame range of su1 kernels as when pollen of TCB Su1/+su1 males is putonto TCB Su1/+su1 females. The latter outcome, considering the 6% ofrecombination between tcb1 and su1 loci, is consistent with completeexclusion of tcb1 pollen. Likely explanations for attentuation of Tcb1observed when the backcross plants were the female parent are that oneor more modifier genes were lost during development of the Ts5 Tcb1stock from TCB or that Ts5 itself dampens Tcb1 action.

A second attentuated Tcb1 strain was identified after repeatedbackcrossing of TCB to inbred W22. The derived Tcb1 strain was comparedwith TCB/+ and Ga1-s/− genotypes for ability to prevent seed set whenwind pollinated with ga1 tcb1 (Table 3, below). Under condition of thistest, Ga1-s/Ga1-s plants were virtually barren and even Ga1-s/ga1heterozygotes produced less than 0.1% set. Not a single kernel set onthe 47TCB/+ plants. In contrast, a 32% set was obtained on attenuatedTcb1 homozygotes and 43% on heterozygotes. This outcome indicates thatinbred W22 carries one or more modifiers that decrease effectiveness ofTcb1 in rejecting Tcb1 pollen. TABLE 3 Average seed sets on plantsdiffering in Ga1:ga1 and Tcb1:tcb1 constitution. Detasseled plants wereallowed to wind pollinate with ga1 tcb1. Average Seed Set Genotype ofFemale Parent No. of Plants (%) ga1 tcb1/ga1 tcb1 71 98.3 Gal-stcb1/Gal-s tcb1 43 0.0^(a) Gal-s tcb1/ga1 tcb1 45 0.1 ga1 Tcb1/gal1 Tcb142 31.9 ga1 Tcb1/ga1 tcb1 45 42.9 TCB/+ 47 0.0^(b)^(a)total of two kernels^(b)zero kernels

Separation of Tcb1 from Ga1-m, present together in the TIC haplotype,provides material suitable for testing interaction between the Ga1-s andTcb1 incompatibility systems. To this end, ga1 Tcb1 or Ga1-s tcb1 pollenwas mixed with ga1 Tcb1 and applied to silks of various incompatabilitygenotypes. The ga1 tcb1 strain used confers colored kernels, the otherstrains colorless. Hence the ga1 tcb1 pollen serves as a tracer todetermine how efficiently the various female parents discriminatebetween ga1 tcb1 and Ga1-s or Tcb1-containing pollen.

Mixtures of ga1 Tcb1 with ga1 tcb1 pollen produced essentially a fullset of kernels on Tcb1-containing ear parents (FIG. 5A), as expectedbased on mixtures of TCB with ga1 tcb1 (FIG. 2D). TCB/+ femalesdiscriminated almost completely against ga1 tcb1 pollen, whereasattentuated Tcb1 homozygotes and heterozygotes (both homozygous gal)averaged approximately 20% as many colored kernels as on compatible ga1tcb1. (See Example 1a for calibrating the proportion of viable pollen ofthe two classes in mixtures.) Ga1-m tcb1/ga1 Tcb1 double heterozygotesplot with the attenuated Tcb1 genotypes rather than with TCB/+. Thus thestrong barrier of TCB is not due to the combination of Tcb1 with Ga1-mas such, but due to enhancement by still other factors. Homozygous Ga1-stcb1 plants pollinated with this mixture were essentially barren.However, Ga1-s plants heterozygous with either gal or Ga1-m (bothhomozygous tcb1) produced partial sets. That there were only about halfas many colored kernels as on fully compatible ga1 tcb1 shows a decidedpreference of Ga1-s/− pistils for Tcb1 over tcb1 pollen.

Mixtures of Ga1-s tcb with ga1 tcb pollen (FIG. 5B) produced good setsof seed on Ga1-s homozygotes and heterozygotes (all tcb1/tcb1) withalmost complete discrimination against ga1 Tcb1 pollen, also asexpected. TCB/+ females pollinated with the mix were almost barren,whereas attentuated Tcb1/tcb1 heterozygotes produced fairly well setears. Neither homozygosity of the attenuated Tcb1 stock nor addition ofGa1-m caused the level of incompatibility to approach that of TCB/+.That there was a smaller fraction of colored kernels on Tcb1/− femalesrelative to ga1 tcb1 again indicates partial cross recognition betweenthe two incompatability systems.

Tcb1 could be expressed either before or after fertilization. Ifpostzygotic, a reduced set of seed should accompany instances ofdistorted segregation, such as the deficit of sugary kernels among F2populations of TCB/su1 heterozygotes. No reduction in set has beenobserved in this circumstance, providing evidence against a post-zygoticmechanism.

Conceivably, however, barrenness observed following other types ofcrosses could reflect postzygotic lethality. To address thispossibility, ten plants in a backcross progeny segregating forheterozygous TCB and standard tcb1/tcb1 plants were pollinated onsuccessive days. Color-marked ga1 tcb1 was applied on day one followedby the plant's own pollen on day two. Six plants produced ears with afull set of mostly colored kernels, indicating compatibility with gal .Four plants produced mostly or only colorless and weakly colored kernelscharacteristic of the TCB/+ parent, indicating maintenance of ovuleviability despite prior pollination with incompatible gal tcbl.

EXAMPLE 1f Location of a Major Modifier of Tcb Action

To locate any modifiers linked to tcb, a Ga1-m Tcb1 Su1 strain havingstrong Tcb action was crossed twice to ga1 Tcb1 su1. Among 62 Tcb1carrying progeny, all but four produced 7.0% or fewer sugary kernelswhen self-pollinated, indicating strong Tcb1 action. The remaining fourears carried from 9.8 to 19.6% sugary, indicating loss of a modifierlocated approximately 6.5 centiMorgans from Tcb. That three of the fouracquired ga1 from ga1 tcb1 su1 parent places the modifier proximal toTcb1, toward ga1. This modifier evidently was removed when Ts5 wasrecombined with Tcb1 to produce the Ts5/Tcb1 test chromosome used to formapping Tcb1. Its absence accounts for attenuation of Tcb1 action in theresulting stock and likewise with the strain extensively backcrossed toW22 reported in Table 3.

EXAMPLE 2 Transfer of the Teosinte Crossing Barrier Trait toCommercially Elite Hybrids and Inbred Lines

Corn plants exhibiting the TCB trait prevent hybridization with certaingenetic tester stocks by rejecting their pollen. This barrier tocrossing can serve to isolate varieties containing TCB trait fromcontamination by unwanted sources of pollen. To test whether TCB wouldprevent pollination by commercially elite hybrids and inbred lines,hybridization experiments were conducted during the 1999/2000 winterseason in test plots located on the Hawaiian island of Molokai.

As shown below in Table 4, pollen from plants of 87 different commercialand test hybrids (see rows 1-110) was placed on silks of a dent inbredW22-TCB heterozygous strain. In one attempted cross per hybrid examinedto date, 74 potential ears bore no kernels, 5 ears had a single kerneland 6 ears had two kernels and 2 ears had 3 kernels. In parallel, pollenof plants representing 21 inbred lines was placed on a W22-TCBhomozygous strain (containing both the Tcb locus and at least onegene(s) which encode for the silk and pollen effect functions, andmodifiers to the Tcb locus in both chromosomes). This data demonstratesthat the teosinte crossing barrier to crossing is effective inpreventing hybridization by commercially elite inbreds and hybrids.TABLE 4 Hawaii Nursery Row Poll² Entry Pollinator Number of # FloweringData #¹ Instruction Code³ Source Row #⁴ Kernels⁵ Plant⁶ Female⁷ Male⁸Source⁹ Notes 1 0+ 50 2, 3, 4, 5, 6, 10 * 7 Jerry Kermicle 2 0−> 701 0 45 FFR Coop 3 0−> 704 0 4 5 FFR Coop 4 0−> 705 0 4 5 FFR Coop 5 0−> 706 05 5 FFR Coop 6 0−> 707 2 5 5 FFR Coop 7 0+ 50 8, 18, 14, 15 * 5 JerryKermicle 8 0−> 708 0 5 5 FFR Coop 9 0−> 711 0 5 6 FFR Coop 10 0−> 712 04 4 FFR Coop 11 0−> 713 * 6 6 FFR Coop 12 0−> 716 0 5 5 FFR Coop 13 0+50 9, 12, 17 * 8 Jerry Kermicle 14 0−> 717 0 7 8 FFR Coop 15 0−> 718 1 99 FFR Coop 16 0−> 719 * 9 9 FFR Coop 17 0−> 720 0, 0 8 9 FFR Coop 18 0−>721 2 9 9 FFR Coop 19 0+ 50 17, 20, 21, 22, 23, 24 * 8 Jerry Kermicle 200−> 723 0 7 8 FFR Coop 21 0−> 724 0 7 9 FFR Coop 22 0−> 725 0 7 9 FFRCoop 23 0−> 727 0 7 8 FFR Coop 24 0−> 728 0 6 7 FFR Coop 25 0+ 60 26,27, 28, 29, 30 * 5 Jerry Kermicle 26 0−> 733 0 7 7 FFR Coop 27 0−> 734 07 7 FFR Coop 28 0−> 735 0 7 8 FFR Coop 29 0−> 736 0 7 8 FFR Coop 30 0−>737 0 7 8 FFR Coop 31 0+ 60 32, 33, 34 * 3 Jerry Kermicle 32 0−> 738 0 89 FFR Coop 33 0−> 739 0 9 9 FFR Coop 34 0−> 740 0 8 9 FFR Coop 35 0−>741 0 9 9 FFR Coop 36 0−> 742 0 9 9 FFR Coop 37 0+ 60 38, 39 * 4 JerryKermicle 38 0−> 743 0 7 7 FFR Coop 39 0−> 744 1 6 6 FFR Coop 40 0−> 7450 7 8 FFR Coop 41 0−> 748 0 6 7 FFR Coop 42 0−> 749 * 7 8 FFR Coop 43 0+60 44 * 1 Jerry Kermicle 44 0−> 750 0 8 7 FFR Coop 45 0−> 751 0 8 8 FFRCoop 46 0−> 752 0 8 9 FFR Coop 47 0−> 753 0 7 7 FFR Coop 48 0−> 754 0 77 FFR Coop 49 0+ 50 50, 51, 52, 53 * Jerry Kermicle 50 0−> 903 0 7 7 FFRCoop 51 0−> 904 0 7 7 FFR Coop 52 0−> 905 0 7 7 FFR Coop 53 0−> 906 0 78 FFR Coop 54 0−> 907 0 7 8 FFR Coop 55 0+ 50 54, 56, 57, 58, 59, 60,65, 66, Jerry Kermicle 70, 71, 83, 84 56 0−> 908 0 7 9 FFR Coop 57 0−>909 0 8 9 FFR Coop 58 0−> 910 0 8 9 FFR Coop 59 0−> 911 0 FFR Coop 600−> 913 2 7 7 FFR Coop 61 0+ 60 62, 63, 64 * Jerry Kermicle 62 0−> 914 07 7 FFR Coop 63 0−> 915 0 7 8 FFR Coop 64 0−> 916 0 9 10 FFR Coop 65 0−>917 0 9 9 FFR Coop 66 0−> 918 2 7 7 FFR Coop 67 0+ 60 68, 69 * JerryKermicle 68 0−> 919 0 9 9 FFR Coop 69 0−> 920 0 10 10 FFR Coop 70 0−>921 0 9 9 FFR Coop 71 0−> 922 0 8 10 FFR Coop 72 0−> 923 * 7 7 FFR Coop73 0+ 60 74, 75, 76, 77, 78 * Jerry Kermicle 74 0−> 924 0 8 8 FFR Coop75 0−> 925 1 6 7 FFR Coop 76 0−> 926 0 8 8 FFR Coop 77 0−> 927 2 8 9 FFRCoop 78 0−> 928 0 7 7 FFR Coop 79 0+ 60 80, 81, 82 * Jerry Kermicle 800−> 929 0 7 8 FFR Coop 81 0−> 930 3 8 9 FFR Coop 82 0−> 931 0 8 8 FFRCoop 83 0−> 932 0 7 8 FFR Coop 84 0−> 933 0 7 8 FFR Coop 85 0+ 60 86 * 1Jerry Kermicle 86 0−> 934 0 7 9 FFR Coop 87 0−> 935 0 7 8 FFR Coop 880−> 936 0 7 8 FFR Coop 89 0−> 937 0 8 8 FFR Coop 90 0−> 938 0 7 7 FFRCoop 91 0+ 50 92, 93, 94 * 2 Jerry Kermicle 92 0−> 939 0 6 7 FFR Coop 930−> 940 0 8 8 FFR Coop 94 0−> 941 1 7 8 FFR Coop 95 0−> 942 0, 1 8 8 FFRCoop 96 0−> 944 2 6 7 FFR Coop 97 0+ 50 89 * 2 Jerry Kermicle 98 0−>2001 0 6 6 Renk Seeds 99 0−> 2002 3 4 4 Renk Seeds 100 0−> 2003 * 4 5Renk Seeds 101 0−> 2004 0 4 4 Renk Seeds 102 0−> 2005 0 6 7 Renk Seeds103 0+ 60 87, 88, 90, 95 * 7 Jerry Kermicle 104 0−> 2006 0 5 6 RenkSeeds 105 0−> 2007 0 5 5 Renk Seeds 106 0−> 2008 0 5 5 Renk Seeds 1070−> 2009 0 6 6 Renk Seeds 108 0−> 2010 0 5 6 Renk Seeds 109 0−> 60 95,96, 101, 102 * 10 Jerry Kermicle 110 0−> 60 98, 99, 104, 105, 106, * 13Jerry Kermicle 107, 108 111 0−> 70 4 Jerry Kermicle 112 0−> 70 3 JerryKermicle 113 0−> 70 9 Jerry Kermicle 114 0−> or 0+ 70 8 Jerry Kermicle115 0−> or 0+ 1 10 Jerry Kermicle 116 0−> or 0+ 1 12 Jerry Kermicle 1170−> or 0+ 1 12 Jerry Kermicle 118 0−> or 0+ 1 18 Jerry Kermicle 119 0−>or 0+ 1 17 Jerry Kermicle 120 0−> and 0+ 3 2 Jerry Kermicle x 121, nk¹⁰121 0−> and 0+ 2011 Inbred A FFR Coop, Inc. 2 x's, x 122 (2) 122 0−> and0+ 3 3 Jerry Kermicle x 123, nk 123 0−> and 0+ 2012 Inbred B FFR Coop,Inc. x, x 159, x 122 (2), x 160 124 0−> and 0+ 3 6 Jerry Kermicle x 125,nk 125 0−> and 0+ 2013 Inbred C 7 8 FFR Coop, Inc. 2x, x 124 (2), x 159,x 160 126 0−> and 0+ 3 5 Jerry Kermicle x 127 (5), 4 nk, 1 k¹¹ 127 0−>and 0+ 2014 Inbred D FFR Coop, Inc. x 160, x 159, 2x, x 126 (6) 128 0−>and 0+ 3 6 Jerry Kermicle x 129 (3), 2 nk, 1k 129 0−> and 0+ 2015 InbredE 7 8 FFR Coop, Inc. x 128, x 159 130 0−> and 0+ 3 7 Jerry Kermicle x131, 2 nk, 1k 131 0−> and 0+ 2016 Inbred F FFR Coop, Inc. x 159, x 160,2x, x 130 (2) 132 0−> and 0+ 3 6 Jerry Kermicle x 132, 2 nk 133 0−> and0+ 542 Inbred G B14 7 8 Renk Seeds 2x, v.v.¹² 134 0−> and 0+ 4 3 JerryKermicle 135 0−> and 0+ 2017 Inbred H IODENT 7 7 Renk Seeds 2x, x 160, x134 (5), x 159 136 0−> and 0+ 4 4 Jerry Kermicle 137 0−> and 0+ 2018Inbred I B14 5 6 Renk Seeds 2x, failed to pollinate 138 0−> and 0+ 4 3Jerry Kermicle 139 0−> and 0+ 2019 Inbred J 3737X 7 7 Renk Seeds 2x, x159 140 0−> and 0+ 4 3 Jerry Kermicle x 139, 1k 141 0−> and 0+ 2020Inbred K B14 7 7 Renk Seeds x 160, 2x, x 159 142 0−> and 0+ 4 2 JerryKermicle x 143, nk 143 0−> and 0+ 2021 Inbred L O43, W117 6 6 Renk Seedsx 160, 2x, x 159 144 0−> and 0+ 4 3 Jerry Kermicle x 145, nk 145 0−> and0+ 2022 Inbred M Renk Seeds x 160, 3x, x144 (2F), x 159 146 0−> or 0+ 26 Jerry Kermicle 147 0−> or 0+ 2 6 Jerry Kermicle 148 0−> or 0+ 2 10Jerry Kermicle 149 0−> or 0+ 2 6 Jerry Kermicle 150 0−> or 0+ 2 8 JerryKermicle 151 0−> or 0+ 2 4 Jerry Kermicle 152 0−> or 0+ 2 8 JerryKermicle 153 0−> or 0+ 70 4 Jerry Kermicle 35 154 0−> or 0+ 70 4 JerryKermicle 36, 40, 41 155 0−> or 0+ 70 4 Jerry Kermicle 45, 46, 47 156 0−>or 0+ 70 2 Jerry Kermicle 48 157 0−> or 0+ 70 2 Jerry Kermicle 182 1580−> or 0+ 70 3 Jerry Kermicle 183 159 0−> or 0+ 2023 4557 x gives 3% suJerry Kermicle 160 0−> or 0+ 2024 Jerry Kermicle 182 0−> and 0+ 2046Inbred N 7 7 Curry Hybrids nk 183 0−> and 0+ 2047 Inbred N 7 7 CurryHybrids nk 184 0−> and 0+ 2048 Inbred O 10 10 Curry Hybrids 2x, x148, x160 185 0−> and 0+ 2049 Inbred O 10 10 Curry Hybrids x 159, x148 186 0−>and 0+ 2050 Inbred P 10 10 Curry Hybrids x 159, x 148, x 160 (20k), 2x187 0−> and 0+ 2051 Inbred P 10 10 Curry Hybrids x 148 188 0−> and 0+2052 Inbred Q 7 7 Curry Hybrids 2x, 148 (nk), x 159, x 160 189 0−> and0+ 2053 Inbred Q 7 7 Curry Hybrids 2x, x 148(25x) 190 0−> and 0+ 2054Inbred R 8 8 Curry Hybrids x 148, x 160(5k), 2x 191 0−> and 0+ 2055Inbred R 8 8 Curry Hybrids x 148, 159 192 0−> and 0+ 2056 Inbred S 8 8Curry Hybrids x 148, 2x 193 0−> and 0+ 2057 Inbred S 8 8 Curry Hybrids x159, x 160, x 148 194 0−> and 0+ 2058 Inbred T 12 12 Curry Hybrids 2x, x148, x 159 195 0−> and 0+ 2059 Inbred T 12 12 Curry Hybrids 2x, x 148196 0−> and 0+ 2060 Inbred U 11 11 Curry Hybrids 2x, x 160, x 148, x 159197 0−> and 0+ 2061 Inbred U 11 11 Curry Hybrids 2x, x 148 198 0−> and0+ Sweet 199 0−> and 0+ Sweet 200 0−> and 0+ Sweet¹Row # = The order in which planted.²Poll = Instruction for whether the plant was used as male or female.³Entry Code = The company designation either for hybrid or inbred.⁴Pollinator Source = Row number from which the male pollen was taken.⁵Number of Kernels = Number of kernels that were pollinated onreciprocal test cross ears.⁶# Plant = Number of plant stand count.⁷Flowering Data - Female = The day in the month that silk had expressedon 50 percent of the ears on the center spike approximately 3 inches.⁸Flowering Data - Male = The day in the month that the pollen hadexpressed on 50 percent of the tassels on the center spike approximately3 inches.⁹Source = The person and/or company that supplied the seed.¹⁰nk = no kernels.¹¹k = number of kernels.¹²v.v. = vivevipary.* = failed to pollinate.

All references cited herein are hereby incorporated by reference.

The present invention is illustrated by way of the foregoing descriptionand examples. The foregoing description is intended as a non-limitingillustration, since many variations will become apparent to thoseskilled in the art in view thereof. It is intended that all suchvariations within the scope and spirit of the appended claims beembraced thereby.

Changes can be made to the composition, operation and arrangement of themethod of the present invention described herein without departing fromthe concept and scope of the invention as defined in the followingclaims.

1. A cross-incompatible maize plant comprising a TCB trait.
 2. Thecross-incompatible plant of claim 1 wherein said plant fails to set seedwhen pollinated by plants lacking the TCB trait but sets seed whenpollinated by plants carrying the TCB trait.
 3. The cross-incompatibleplant of claims 1 or 2 wherein said plant maintains functional pollenand sets seed when pollinated by itself or causes other maize plants toset seed when pollinated by said plant.
 4. The cross-incompatible maizeplant of claim 1 wherein said maize plant is an inbred plant.
 5. Thecross-incompatible maize plant of claim 1 wherein said maize plant is ahybrid plant.
 6. The cross-incompatible maize plant of claim 1 whereinsaid maize plant is a haploid plant.
 7. The cross-incompatible maizeplant of claim 1 wherein said maize plant is an apomictic maize plant.8. The cross-incompatible maize plant of claims 1, 4, 5, 6 or 7 whereinsaid plant is a genetically engineered plant.
 9. The cross-incompatiblemaize plant of claim 9 further comprising a gene cluster within itsgenome wherein said gene cluster is located on the short arm ofchromosome 4 between map units 40-85.
 10. The cross-incompatible maizeplant of claim 9 further comprising a Tcb locus within its genome. 11.The cross-incompatible maize plant of claim 10 wherein said Tcb locus islocated on the short arm of chromosome 4 about 6 map units distal to thesugary1 gene and about 40 map units from the Ga1gene.
 12. Thecross-incompatible maize plant of claim 10 wherein said Tcb locuscomprises at least one gene which encodes for a silk effect function insaid plant.
 13. The cross-incompatible maize plant of claims 10 or 12wherein said Tcb locus comprises at least one gene which encodes for apollen effect function in said plant.
 14. The cross-incompatible maizeplant of claims 9, 10, 12 or 13 further comprising at least one modifiergene within its genome.
 15. A cross-incompatible maize plant comprisinga TCB trait and which (1) fails to set seed when pollinated by plantslacking the TCB trait but sets seed when pollinated by plants carryingthe TCB trait; and (2) maintains functional pollen and sets seed whenpollinated by itself or causes other maize plants to set seed whenpollinated by said plant.
 16. The cross-incompatible maize plant ofclaim 15 wherein said maize plant is an inbred plant.
 17. Thecross-incompatible maize plant of claim 15 wherein said maize plant is ahybrid plant.
 18. The cross-incompatible maize plant of claim 15 whereinsaid maize plant is a haploid plant.
 19. The cross-incompatible maizeplant of claim 15 wherein said maize plant is an apomictic maize plant.20. The cross-incompatible maize plant of claims 15, 16, 17, 18 or 19wherein said plant is a genetically engineered plant.
 21. Thecross-incompatible maize plant of claim 15 further comprising a genecluster within its genome wherein said gene cluster is located on theshort arm of chromosome 4 between map units 40-85.
 22. Thecross-incompatible maize plant of claim 21 further comprising a Tcblocus within its genome.
 23. The cross-incompatible maize plant of claim22 wherein said Tcb locus is located on the short arm of chromosome 4about 6 map units distal to the sugary1 gene and about 40 map units fromthe Ga1gene.
 24. The cross-incompatible maize plant of claim 22 whereinsaid Tcb locus comprises at least one gene which encodes for a silkeffect function in said plant.
 25. The cross-incompatible maize plant ofclaims 22 or 24 wherein said Tcb locus comprises at least one gene whichencodes for a pollen effect function in said plant.
 26. Thecross-incompatible maize plant of claims 21, 22, 24 or 25 furthercomprising at least one modifier gene within its genome.
 27. Across-incompatible maize plant comprising a TCB trait and wherein saidTCB trait is derived from plant W22-TCB deposited as ATCC No. PTA-1601.28. The cross-incompatible maize plant of claim 27 further comprising agene cluster within its genome wherein said gene cluster is located onthe short arm of chromosome 4 between map units 40-85.
 29. Thecross-incompatible maize plant of claim 28 further comprising a Tcblocus within its genome.
 30. The cross-incompatible maize plant of claim29 wherein said Tcb locus is located on the short arm of chromosome 4about 6 map units distal to the sugary1 gene and about 40 map units fromthe Ga1gene.
 31. The cross-incompatible maize plant of claim 29 whereinsaid Tcb locus comprises at least one gene which encodes for a silkeffect function in said plant.
 32. The cross-incompatible maize plant ofclaims 29 or 31 wherein said Tcb locus comprises at least one gene whichencodes for a pollen effect function in said plant.
 33. Thecross-incompatible maize plant of claims 28, 29, 31 or 32 furthercomprising at least one modifier gene within its genome.
 34. Thecross-incompatible maize plant of claim 27 wherein said maize plant isan inbred plant.
 35. The cross-incompatible maize plant of claim 27wherein said maize plant is a hybrid plant.
 36. The cross-incompatiblemaize plant of claim 27 wherein said maize plant is a haploid plant. 37.The cross-incompatible maize plant of claim 27 wherein said maize plantis an apomictic maize plant.
 38. The cross-incompatible maize plant ofclaims 34, 35, 36 or 37 wherein said plant is a genetically engineeredplant.
 39. A process for obtaining an inbred maize plant, which whencrossed with a second inbred maize plant, produces a hybrid maize plantwhich is cross-incompatible and contains a TCB trait, the processcomprising the steps of: a) selecting a first donor parental maize plantfrom a population of maize plants, wherein said first donor parentalmaize plant is cross-incompatible and contains a TCB trait; b) crossingsaid selected first donor parental maize plant with a second parentalmaize plant containing genes which encode for desirable traits in hybridcombination; c) collecting the seed resulting from the cross in step b);d) planting and growing the seed collected in step c) under plant growthconditions; e) screening the resulting plant population for the presenceof the TCB trait identified in step (a); and f) selecting plants fromsaid population having the TCB trait for cross-incompatibility forfurther crossings and screenings until a line is obtained which ishomozygous for the TCB trait for cross-incompatibility to provide such atrait in an inbred to be used in hybrid combination.
 40. The process ofclaim 39 wherein the first donor parental maize plant further comprisesa gene cluster within its genome wherein said gene cluster is located onthe short arm of chromosome 4 between map units 40-85.
 41. The processof claim 40 wherein the first donor parental maize plant furthercomprises a Tcb locus.
 42. The process of claim 41 wherein said Tcblocus is located on the short arm of chromosome 4 about 6 map unitsdistal to the sugary1 gene and about 40 map units from the Ga1gene. 43.The process of claim 41 wherein said Tcb locus comprises at least onegene which encodes for a silk effect function in said plant.
 44. Theprocess of claims 41 or 43 wherein said Tcb locus comprises at least onegene which encodes for a pollen effect function in said plant.
 45. Theprocess of claims 40, 41, 43 or 44 wherein the first donor parentalmaize plant further comprises at least one modifier gene.
 46. Theprocess of claim 39 wherein the second parental maize plant iscross-incompatible and comprises a TCB trait.
 47. A cross-incompatibleinbred maize plant comprising a TCB trait produced by the process ofclaim
 39. 48. A process for producing a cross-incompatible hybrid maizeplant exhibiting a TCB trait, the process comprising the steps of: a)crossing the inbred maize plant of claim 39 with a second maize inbredline comprising genes encoding desirable phenotypic traits to produce asegregating plant population; and b) collecting the hybrid seedresulting from the cross in step a).
 49. The process of claim 48 whereinthe second maize inbred line is cross-incompatible and comprises a TCBtrait.
 50. A cross-incompatible hybrid maize plant comprising a TCBtrait produced by the process of claim
 48. 51. A process for selecting afirst donor parental maize plant suitable for use in producing an inbredmaize plant, which inbred maize plant, if crossed with a second inbredmaize plant, produces a hybrid maize plant which is cross-incompatibleand contains a TCB trait, the process comprising the steps of: analyzingeach plant from a population of maize plants for the presence of a TCBtrait.
 52. The process of claim 51 further comprising the step ofanalyzing the DNA of each plant from said population for a gene clusterwithin its genome wherein said gene cluster is located on the short armof chromosome 4 between map units 40-85.
 53. The process of claim 52further comprising the step of analyzing the DNA of each plant from saidpopulation for a Tcb locus.
 54. The process of claim 53 wherein said Tcblocus is located on the short arm of chromosome 4 about 6 map unitsdistal to the sugary1 gene and about 40 map units from the gene Ga1gene.55. The process of claim 53 further comprising the step of analyzing theDNA of each plant from said population for at least one gene whichencodes for a silk effect function in said plant.
 56. The process ofclaims 52, 53 or 55 further comprising the step of analyzing the DNA ofeach plant from said population for at least one gene which encodes fora pollen effect function in said plant.
 57. The process of claims 52,53, 55 or 56 further comprising the step of analyzing the DNA of eachplant of said population for at least one modifier gene.
 58. Across-incompatible first donor parental maize plant comprising a TCBtrait produced by the process of claim
 51. 59. A process for selecting across-incompatible hybrid maize plant containing a TCB trait, theprocess comprising the steps of: analyzing each plant from a populationof hybrid maize plants for the a TCB trait.
 60. The process of claim 59further comprising the step of analyzing the DNA of each plant from saidpopulation for a gene cluster wherein said gene cluster is located onthe short arm of chromosome 4 between map units 40-85.
 61. The processof claim 60 further comprising the step of analyzing the DNA of eachplant from said population for a Tcb locus.
 62. The process of claim 61wherein said Tcb locus is located on the short arm of chromosome 4 about6 map units distal to the sugary1 gene and about 40 map units from thegene Ga1gene.
 63. The process of claim 61 further comprising the step ofanalyzing the DNA of each plant from said population for at least onegene which encodes for a silk effect function in said plant.
 64. Theprocess of claims 61 or 63 further comprising the step of analyzing theDNA of each plant from said population for at least one gene whichencodes for a pollen effect function in said plant.
 65. The process ofclaims 60, 61, 63 or 64 further comprising the step of analyzing the DNAof each plant of said population for at least one modifier gene.
 66. Across-incompatible hybrid maize plant comprising a TCB trait produced bythe process of claim
 60. 67. A process of controlling hybridization of amaize plant in a field, the process comprising the step of planting in afield a cross-incompatible maize plant of claims 1, 4, 5, 7, 8, 15, 16,17, 19, 20, 27, 34, 35, 37, 38, 47, 50, 58 or
 66. 68. A process ofcontrolling hybridization of inbred maize plants in a field being usedin hybrid seed production, the process comprising the step of plantingin a field being used for hybrid seed production, a cross-incompatibleinbred maize plant of claims 4, 8, 16, 20, 34, 38, 47 or 58.